Analysis of Compositional Bias in a Commercial Phage Display Peptide Library by Next-Generation Sequencing

Abstract

The principal presumption of phage display biopanning is that the naïve library contains an unbiased repertoire of peptides, and thus, the enriched variants derive from the affinity selection of an entirely random peptide pool. In the current study, we utilized deep sequencing to characterize the widely used Ph.DTM-12 phage display peptide library (New England Biolabs). The next-generation sequencing (NGS) data indicated the presence of stop codons and a high abundance of wild-type clones in the naïve library, which collectively result in a reduced effective size of the library. The analysis of the DNA sequence logo and global and position-specific frequency of amino acids demonstrated significant bias in the nucleotide and amino acid composition of the library inserts. Principal component analysis (PCA) uncovered the existence of four distinct clusters in the naïve library and the investigation of peptide frequency distribution revealed a broad range of unequal abundances for peptides. Taken together, our data provide strong evidence for the notion that the naïve library represents substantial departures from randomness at the nucleotide, amino acid, and peptide levels, though not undergoing any selective pressure for target binding. This non-uniform sequence representation arises from both the M13 phage biology and technical errors of the library construction. Our findings highlight the paramount importance of the qualitative assessment of the naïve phage display libraries prior to biopanning.

Depth of Sequencing Plays a Determining Role in the Characterization of Phage Display Peptide Libraries by NGS

“…To achieve this purpose, a sequencing platform capable of rigorous characterization of the composition and diversity of the peptide pool is crucial. High-throughput sequencing has been found to be suited for the analysis of the sequence composition and inherent diversity of phage display libraries [ 20 , 31 ]. In the current work, we made a side-by-side comparison of the capacity of two NGS platforms with different depths of sequencing in characterizing the composition and diversity of a phage display peptide library.…”

“…The available diversity is calculated by titration of amplified phages after electroporation of the library into the host bacterium. Our previous report indicated that the diversity of the library (used in the current work) can be even lower due to the presence of stop codons, frameshifts, and clones without insert in the naïve library [ 20 ]. Consistent with this, the real diversity of phage display peptide libraries is expected to be within the range of 10 8 –10 9 .…”

“…To process the FASTQ files, a MATLAB script modified from [ 36 ] was used, as described previously in [ 20 , 37 ]. The script can be found at (accessed on 27 January 2023).…”

“…The MATLAB script used to generate stacked bar plots can be found at (accessed on 27 January 2023). Furthermore, an in-house Python script in Jupyter Notebook was used to calculate the positional frequency, one hot encoding was used for the amino acids, as described previously [ 20 ]. This is available at (accessed on 27 January 2023).…”

“…This is valuable for monitoring, troubleshooting, and evaluating the design and construction of unselected naïve libraries, gaining detailed insights into the impact of the selective pressure applied to the library, and obtaining a solid understanding of the evolution of peptide populations during library selection [ 15 ]. NGS has not only been performed on biopanning eluates [ 16 , 17 , 18 ] but has also been exploited to investigate unselected and amplified phage display libraries [ 13 , 14 , 19 , 20 , 21 ]. A pivotal parameter for using NGS is the sequencing depth.…”